Image by Dr. Alexandre Neves
Glioblastoma multiforme (GBM) is the most prevalent and deadly form of brain cancer. In GBM, as well as in other solid tumors, it has been proposed that a small subset of tumor cells endowed with stem cell characteristics is responsible for tumor maintenance and therapeutic resistance. Because stem-like tumor cells exhibit an increase in both their self-renewal ability and expression of stem cell markers, it is conceivable that genes that regulate stem cell identity could represent attractive therapeutic targets. A previous study from Dr. Eric Holland’s Laboratory (Human Biology Division), developed the side population (SP) assay to isolate such stem-like tumors cells from whole tumors. The SP assay is based on the ability of cells to pump out a DNA dye (Hoechst 33342) and was shown to rely on the activity of an ATP-binding cassette transporter called ABCG2. A follow-up study from the Holland Laboratory, recently published in Scientific Reports, used both mouse and human GBM models to address whether ABCG2 simply correlates with stem cell characteristics, or alternatively, if it actively drives stem cell properties in GBM.
First, the authors engineered cells derived from either a mouse model of GBM (PDGF-induced glioma primary cultures; PIGPCs) or a human glioma cell line (U87MG) wherein ABCG2 expression could be manipulated. Overexpression of ABCG2 in U87MG cells led to increased expression of five out of the six stem cell markers tested, and this increase could be reverse by pharmacological inhibition of ABCG2 using Fumitremorgin C (FTC). Similarly, inhibition of ABCG2 expression or activity in PIGPCs decreased expression of stem cell markers both at the mRNA and protein level, with concomitant increase in expression of two differentiation markers (Tuj1 and CNPase). Next, the authors queried the ability of sorted SP cells from both the PIGPC and U87MG systems to form spheres using the extreme limiting dilution assay, an in vitro measure of self-renewal. In all cases tested, ABCG2 activity correlated with sphere formation ability. To test for a role in radiation resistance and tumorigenicity, ABCG2-overexpressing cells were irradiated with 0-10Gy and injected intracranially into nude mice, respectively. Strikingly, both ABCG2-overexpressing cells and tumors appeared indistinguishable from controls, suggesting that ABCG2 did not affect colony formation of irradiated cells, nor did it affect impact tumorigenicity in vivo. Finally, the investigators sought to determine how ABCG2 controls expression of stem cell markers, and they surprisingly found that ABCG2-mediated expression of Hes1, Sox2 and Oct4 in ABCG2-overexpressing PIGPCs occurred independently of the Notch pathway, which is known to be required for stem-like properties of some cancers. Moreover, with the exception of Hes1, all of the ABCG2-induced genes relied on the transcription factor Mef for their expression. In summary, by studying the role of the transporter and drug efflux pump ABCG2 in both mouse and human models of glioma, this work showed that increased self-renewal and expression of stem cell genes can be functionally uncoupled from the more clinically relevant features of radiation resistance and tumor formation.
Wee B, Pietras A, Ozawa T, Bazzoli E, Podlaha O, Antczak C, Westermark B, Nelander S, Uhrbom L, Forsberg-Nilsson K, Djaballah H, Michor F, Holland EC. 2016. ABCG2 regulates self-renewal and stem cell marker expression but not tumorigenicity or radiation resistance of glioma cells. Sci Rep, 6, 25956.
Funding for this work was provided by the National Institutes of Health.
Bleau AM, Hambardzumyan D, Ozawa T, Fomchenco EI, Huse JT, Brennan CW, Holland EC. 2009. PTEN/ PI3K/ Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell, 4(3): 226-35.